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Results for “Reduced Folate Carrier Protein”

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PubMed1995

Distinguishing between folate receptor-alpha-mediated transport and reduced folate carrier-mediated transport in L1210 leukemia cells.

L1210 leukemia cells transport reduced folates and methotrexate via a well defined reduced folate carrier system and, in the absence of low folate selective pressure, do not express an alternate endocytotic route mediated by cell surface folate receptors. This laboratory previously described an L1210 leukemia cell line, MTXrA, with acquired resistance to methotrexate (MTX) due to the loss of mobility of the reduced folate carrier. We now report on the transfection of MTXrA with a cDNA encoding the murine homolog of the human folate receptor isoform of KB cells to produce MTXrA-TF1, which constitutively expresses high levels of FR-alpha. MTXrA-TF1 and L1210 cells were utilized to compare transport of methotrexate mediated by FR-alpha and the reduced folate carrier, respectively. Methotrexate influx in the two lines was similar when the extracellular level was 0.1 microM, but as the methotrexate concentration increased, influx via the reduced folate carrier increased in comparison to influx mediated by FR-alpha. Transport kinetics indicated both a approximately 20-fold lower influx Kb and Vmax for MTXrA-TF1 as compared to L1210 cells. The two cell lines exhibited distinct influx properties. Methotrexate influx in MTXrA-TF1 was markedly inhibited by 50 nM folic acid and metabolic poisons. In L1210 cells, 1.0 microM folic acid did not affect MTX influx, and metabolic poisons either had no effect on or increased methotrexate influx. Removal of extracellular chloride markedly inhibited transport in MTXrA-TF1 but stimulated influx in L1210 cells. When the pH was decreased to 6.2, methotrexate influx was not altered in MTXrA-TF1 but was reduced in L1210 cells. Probenecid and sulfobromophthalein inhibit methotrexate influx in both L1210 and MTXrA-TF1 cell lines; however, inhibition in MTXrA-TF1 could be accounted for on the basis of inhibition of methotrexate binding to FR-alpha. The data indicate that the reduced folate carrier and FR-alpha function independently and exhibit distinct properties. FR-alpha expressed at sufficient levels can mediate influx of MTX and folates into cells at rates comparable to the reduced folate carrier and hence has pharmacologic and physiologic importance.

Alkenes↗

PubMed2004

Antifolate resistance in a HeLa cell line associated with impaired transport independent of the reduced folate carrier.

Prior studies from this laboratory documented the prevalence of methotrexate (MTX) transport activity with a low pH optimum in human solid tumor cell lines. In HeLa cells, this low pH activity has high affinity for pemetrexed [PMX (Alimta)] and is reduced folate carrier (RFC)-independent because it is not diminished in a RFC-null subline (R5). R5 cells also have residual transport activity, with high specificity for PMX, at neutral pH. In the current study, a R5 subline, R1, was selected under MTX selective pressure at a modest reduction in pH. There was markedly decreased MTX and PMX transport at both pH 5.5 and pH 7.4. When MTX was removed, there was a slow return of transport activity, and when MTX was added back, there was loss of transport at both pH values within 8 weeks. In R1 cells, there was a marked decrease in accumulation of PMX, MTX, and folic acid along with a decrease in growth inhibition by these and other antifolates that require a facilitative process to gain entry into cells. These data demonstrate that (i) RFC-independent transport in HeLa cells at low and neutral pH contributes to antifolate activity (in particular, to PMX activity) and can be diminished by antifolate selective pressure and (ii) the loss of these activities results in marked resistance to PMX, an agent for which there is little or no loss of activity when transport mediated by RFC is abolished. These observations suggest that transport activity in RFC-null HeLa R5 cells at neutral and low pH may reflect the same carrier-mediated process.

Antimetabolites, Antineoplastic↗

PubMed2002

Resistance to multiple novel antifolates is mediated via defective drug transport resulting from clustered mutations in the reduced folate carrier gene in human leukaemia cell lines.

We have studied the molecular basis of resistance of multiple human leukaemia CCRF-CEM sublines to the novel antifolates ZD9331, GW1843, AG2034, PT523 and edatrexate, which use the reduced folate carrier (RFC) as their main cellular uptake route and that target different folate-dependent enzymes. Antifolate-resistant sublines established by stepwise and single-step selections displayed up to 2135-fold resistance to the selection drug, and up to 2323-fold cross-resistance to various hydrophilic antifolates. In contrast, these sublines were up to 17- and 20-fold hypersensitive to the lipophilic antifolates AG377 and trimetrexate, respectively. The total reduced folate pool of these antifolate-resistant sublines shrunk by 87-96%, resulting in up to 42-fold increased folic acid growth requirement. These sublines lost 92-97% of parental [(3)H]methotrexate influx rates. Genomic PCR single-strand conformational polymorphism analysis and sequencing revealed that most of these drug-resistant sublines harboured RFC mutations that surprisingly clustered in two confined regions in exons 2 and 3. The majority of these mutations resulted in frame-shift and/or premature translation termination and lack of RFC protein expression. The remaining mutations involved single amino acid substitutions predominantly residing in the first transmembrane domain (TMD1). Some RFC-inactivating mutations emerged during the early stages of antifolate selection and were stably retained during further drug selection. Furthermore, some sublines displayed a markedly decreased or abolished RFC mRNA and/or protein expression. This constitutes the first demonstration of clustering of multiple human RFC mutations in TMD1, thereby suggesting that it plays a functional role in folate/antifolate binding and/or translocation. This is the first molecular characterization of human RFC-associated modalities of resistance to various novel antifolates in multiple leukaemia sublines.

Base Sequence↗

PubMed1997

Characterization of N5-methyltetrahydrofolate uptake in cultured human retinal pigment epithelial cells.

PURPOSE: To determine the identity of the transport process that is responsible for the uptake of N5-methyltetrahydrofolate, the predominant form of folate in blood, into cultured human retinal pigment epithelial cells. METHODS: Human retinal pigment epithelial cells were cultured on an impermeable plastic support, and the characteristics of the uptake of radiolabeled N5-methyltetrahydrofolate into the cells were investigated. The expression of the folate receptor and the reduced-folate transporter in these cells was evaluated by functional assays and by Northern blot analysis. In addition, the characteristics of N5-methyltetrahydrofolate uptake in these cells were compared with those in folate transport-defective human breast cancer cells that were manipulated to express functionally by transfection the cloned human folate receptor and the human reduced-folate transporter. RESULTS: Transport of N5-methyltetrahydrofolate into these cells occurred by a single saturable process with a Michaelis-Menten constant of 0.13 +/- 0.01 microM. The process was specific for such reduced folates as N5-methyltetrahydrofolate and N5-formyltetrahydrofolate. Nonreduced folate interacted with this transport process only weakly. The transport process was inhibited by anion transport inhibitors. Results of Northern blot analysis indicated the presence of transcripts specific for the reduced-folate transporter in these cells. These cells expressed very small amounts of the folate receptor evidenced from the binding of folate and from the detectable presence of folate receptor-specific transcript, but there was no evidence for participation of the receptor in the observed transport of N5-methyltetrahydrofolate. There was also no evidence in these cells for expression of the folate transporter that prefers nonreduced folate as a substrate. CONCLUSIONS: Transport of N5-methyltetrahydrofolate in human retinal pigment epithelial cells occurs exclusively through the reduced-folate transporter. The folate receptor is expressed at negligible levels in these cells and does not participate in the observed transport. Because the cells were cultured on impermeable supports, making the basolateral membrane inaccessible for transport measurements, it is speculated that the observed findings are related to the transport function of the apical membrane of these polarized cells.

Carrier Proteins↗

PubMed2006

Absorption of folate by Caco-2 cells is not affected by high glucose concentration.

The aim of this work was to investigate the effect of high glucose exposure on the absorption of folate by Caco-2 cells. We verified that apical high glucose did not affect the apical uptake of [(3)H]folate. Both different concentrations of glucose (10-45 mM) and different exposure times (10 min-24 h) were tested. Furthermore, apical high glucose (30 mM) did not affect the intracellular steady-state levels of [(3)H]folate, and simultaneous apical and basolateral high glucose (30 mM) did not change the apical-to-basolateral apparent permeability (P(app)) to [(3)H]folate. Both the apical uptake and the steady-state intracellular levels of [(3)H]folate were strongly reduced by 5-methyltetrahydrofolate, methotrexate, SITS (4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulfonic acid), DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) and indomethacin, but were not affected or only hardly affected by p-aminohippuric acid and fumitremorgin C. Moreover, DIDS and indomethacin significantly reduced (by 50-60%) the apical-to-basolateral P(app) to [(3)H]folate, but [(3)H]folate present in the cells at the end of the experiment was higher in the case of indomethacin. Fumitremorgin C had no effect. The effect of the drugs tested was not changed or only hardly changed by high glucose. In conclusion, absorption of [(3)H]folate is not modulated by either apical or basolateral high glucose exposure in Caco-2 cells. Moreover, our results suggest that the apical uptake of [(3)H]folate by Caco-2 cells involves the Reduced Folate Transporter (but not the Organic Anion Transporter), and that Multidrug Resistance Protein and/or Organic Anion Transporter (but not Breast Cancer Resistance Protein) may mediate apical efflux of [(3)H]folate.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2006

Molecular basis for decreased folylpoly-gamma-glutamate synthetase expression in a methotrexate resistant CCRF-CEM mutant cell line.

A CCRF-CEM mutant, CEM-p, has been shown to exhibit resistance to methotrexate due to decreased methotrexate polyglutamate accumulation. To ascertain the mechanism(s) responsible for this phenotype, we analyzed FPGS and SLC19A1 mRNA expression, examined FPGS promoter activity, and determined nucleotide sequence of the FPGS promoter and full length cDNA from CCRF-CEM and CEM-p cells. We identified in FPGS from CEM-p cells three amino acid substitutions that altered the ATP binding P-loop, glutamate/folate binding, and a conserved domain located at the carboxyl-terminal. Our data demonstrated for the first time the importance of the highly conserved domain (VTGSLHLVGGV) located at the carboxyl-terminal for FPGS activity.

Amino Acid Sequence↗

PubMed2000

Cloning and functional characterization of the bile acid-sensitive methotrexate carrier from rat liver cells.

We have cloned two complementary DNAs (cDNAs), RL-Mtx-1 and RL-Mtx-2, corresponding to the bile acid- sensitive methotrexate carrier from rat liver by direct full-length rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) using degenerated primers that were deduced from published sequences of tumor cell methotrexate transporters. When expressed in Xenopus laevis oocytes and cosM6 cells, both clones mediate methotrexate and bumetanide transport. RL-Mtx-1 consists of 2,445 bp with an open reading frame of 1,536 bp. The corresponding protein with 512 amino acids has a molecular weight of 58 kd. RL-Mtx-2 (2,654 bp) differs by an additional insert of 203 bp. This insert is located in frame at position 1,196 of the RL-Mtx-1 and contains the typical splice junction sites at the 5' and 3' end, indicating that the RL-Mtx-2 messenger RNA (mRNA) is generated by alternative splicing. The insert contains a stop codon that shortens the RL-Mtx-2 protein to 330 amino acids (38 kd). Both cDNAs contain the binding site sequence for the dioxin/nuclear translocator responsive element (Ah/Arnt-receptor) in conjunction with a barbiturate recognition sequence (Barbie box). Preliminary results show that the Barbie box acts as a negative regulatory element. The two liver cDNA clones show homologies to the published sequences of folate and the reduced folate carriers, but no homology is found to the transport systems for organic anions like the Ntcp1, oatp1, OAT-K1, and OAT1. Expression of the mRNA for the methotrexate carrier is found in liver, kidney, heart, brain, spleen, lung, and skeletal muscle, but not in the testis as revealed by Northern blot analysis. The highest abundance of the mRNA is found in the kidney.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed1999

Mechanisms of methotrexate resistance in osteosarcoma.

High-dose methotrexate is a major component of current protocols for the treatment of osteosarcoma, but some tumors seem to be resistant. Potential mechanisms of resistance include decreased transport through the reduced folate carrier (RFC) and increased expression of dihydrofolate reductase (DHFR). To investigate methotrexate resistance, tumors were obtained from 42 patients with high-grade osteosarcoma. RFC and DHFR mRNA expression were studied by semiquantitative reverse transcription-PCR. The RFC and DHFR genes were studied for deletions and amplification by Southern blot. Thirteen of 20 (65%) osteosarcoma samples were found to have decreased RFC expression at the time of initial biopsy. At definitive surgery and relapse, 10 of 22 (45%) were found to have decreased RFC expression. Seventeen of 26 (65%) samples with a poor response to chemotherapy had decreased RFC expression, whereas 5 of 14 (36%) samples with a good response had a decrease (P = 0.03). None of the samples had an RFC gene deletion. Two of 20 samples (10%) showed increased DHFR expression at initial biopsy. The frequency of increased DHFR expression was significantly higher in metastatic or recurrent tumors (62%, P = 0.014). None of the samples showed evidence of DHFR gene amplification. The high frequency of decreased RFC expression in the biopsy material suggests that impaired transport of methotrexate is a common mechanism of intrinsic resistance in osteosarcoma. Increased DHFR expression in the pulmonary metastases may be a mechanism of acquired methotrexate resistance or a difference between primary and metastatic lesions.

Adolescent↗

PubMed2002

The murine-reduced folate carrier gene can act as a selectable marker and a suicide gene in hematopoietic cells in vivo.

Increased expression of the reduced folate carrier confers sensitivity to the antifolate drug methotrexate because it results in increased cellular uptake of this drug, and increased resistance to trimetrexate, a lipid-soluble antifolate drug, because it enables cells to take up exogenous folates that rescue cells from antifolate cytotoxicity. We therefore hypothesized that the reduced folate carrier could act as a suicide gene after treatment with methotrexate and as a selectable marker after exposure to trimetrexate. To test this hypothesis, we constructed replication-defective retroviruses containing the murine-reduced folate carrier (mRFC). Murine bone marrow cells transduced with the mRFC-containing retrovirus showed increased sensitivity to methotrexate and increased resistance to trimetrexate compared to empty vector-transduced controls in colony forming assays. Furthermore, colonies surviving trimetrexate and methotrexate treatment showed an enrichment of the mRFC gene after exposure to trimetrexate and a decrease after exposure to methotrexate. Lethally irradiated mice transplanted with bone marrow cells transduced with the mRFC-retrovirus and treated with the antifolate drugs after hematopoietic recovery demonstrated a relative increase in the number of cells containing the mRFC transgene after trimetrexate treatment and a decrease after methotrexate treatment. Therefore, these studies demonstrate the potential of the reduced folate carrier gene to play a dual role in gene therapy applications.

Animals↗

PubMed2004

[Advances in the study of the etiologic relationship between reduced folate carrier gene (RFC1) and neural tube and craniofacial defects].

Neural tube and craniofacial defects are common birth defects which are ascribed to the combination of genetic and environmental factors. The population epidemiological studies suggested that periconceptional use of multivitamins containing folic acid can reduce a woman's risk of having a child with neural tube and craniofacial defects. It's a major environmental factor that periconceptional women with deficiency of folic acid may increase their risk for delivering babies with neural tube and craniofacial defects, but the mechanism by which folic acid facilitates this risk reduction is unknown. This paper reviews folate transport carrier, Reduced Folate Carrier (RFC)'s characteristics in biological chemistry, physiological function, the folate transport mechanism, structure, function, regulation and expression of reduced folate carrier gene (RFC1), and the relationship between RFC1 with plasm or erythrocyte folate level and neural tube defects, et al. It is suggested a etiologic hypothesis in investigation of candidate gene encoding specific folate-related pathways of neural tube and craniofacial defects.

Carrier Proteins↗

PubMed2004

The H(+)-dependent reduced folate carrier 1 of humans and the sodium-dependent methotrexate carrier-1 of the rat are orthologs.

Previously, two different carrier systems for uptake of reduced folates and the antifolate methotrexate (Mtx) were described: the pH-dependent folate sensitive reduced folate carrier 1 (RFC1) from human, hamster and mouse and a sodium-dependent and folate insensitive Mtx carrier-1 (MTX-1) from rat. It was found that all critical residues of the homologous amino acid sequence were identical. RFC1- as well as MTX-1-mediated uptake of a marker substrate into suitable human and rat cell lines increased with proton concentration, was sodium-dependent at neutral pH, and inhibited by folate at acidic pH. It is concluded that RFC1 and MTX-1 are orthologs.

Amino Acid Sequence↗

PubMed2005

Reduced folate carrier gene is a risk factor for neural tube defects in a Chinese population.

BACKGROUND: There is a considerable body of data demonstrating that periconceptional supplementation of folic acid can prevent a significant proportion of neural tube defects (NTDs). At present, the mechanism by which folic acid exerts its beneficial effect remains unknown. Folate transporter genes, including the reduced folate carrier gene (RFC1), have been proposed as NTD risk factors. METHODS: The study population included 104 nuclear families with NTDs and 100 nonmalformed control families. We investigated the possible association between a common RFC1 polymorphism (A80G) and NTD risk among offspring, as well as potential gene-environment interactions between the infant RFC1 genotype and maternal periconceptional use of folic acid through a population-based case-control study. RESULTS: We observed that the infants of the GG genotype were associated with a 2.56-fold increased risk of NTDs when compared to the AA genotype (odds ratio [OR], 2.56; 95% confidence interval [CI], 1.04-6.36) in our study population. Among mothers who did not utilize folic acid supplements, the risk for having a child with an NTD was 3.30 (95% CI, 1.15-9.65) for offspring with the GG genotype, compared to the reference (AA) genotype. Children who had the GG genotype and whose mothers did not take folic acid had an elevated risk for NTDs (OR, 8.80; 95% CI, 2.83-28.69), compared to offspring with the AA and GA genotypes whose mothers utilized folic acid supplements. CONCLUSIONS: Our findings suggest that the RFC1 G allele is likely to be an important genetic factor in determining folate transport and subsequently may be a risk factor for NTDs in this Chinese population.

Case-Control Studies↗

PubMed2004

Characterization of a folate transporter in HeLa cells with a low pH optimum and high affinity for pemetrexed distinct from the reduced folate carrier.

Studies were undertaken to characterize a low pH transport activity in a reduced folate carrier (RFC)-null HeLa-derived cell line (R5). This transport activity has a 20-fold higher affinity for pemetrexed (PMX; Kt, approximately 45 nmol/L) than methotrexate (MTX; Kt, approximately 1 micromol/L) with comparable Vmax values. The Ki values for folic acid, ZD9331, and ZD1694 were approximately 400-600 nmol/L, and the Ki values for PT523, PT632, and trimetrexate were >50 micromol/L. The transporter is stereospecific and has a 7-fold higher affinity for the 6S isomer than the 6R isomer of 5-formyltetrahydrofolate but a 4-fold higher affinity for the 6R isomer than the 6S isomer of dideazatetrahydrofolic acid. Properties of RFC-independent transport were compared with transport mediated by RFC at low pH using HepG2 cells, with minimal constitutive low pH transport activity, transfected to high levels of RFC. MTX influx Kt was comparable at pH 7.4 and pH 5.5 (1.7 versus 3.8 micromol/L), but Vmax was decreased 4.5-fold. There was no difference in the Kt for PMX (approximately 1.2 micromol/L) or the Ki for folic acid (approximately 130 micromol/L) or PT523 ( approximately 0.2 micromol/L) at pH 7.4 and pH 5.5. MTX influx in R5 and HepG2 transfectants at pH 5.5 was trans-stimulated in cells loaded with 5-formyltetrahydrofolate, inhibited by Cl- (HepG2-B > R5), Na+ independent, and uninhibited by energy depletion. Hence, RFC-independent low pH transport activity in HeLa R5 cells is consistent with a carrier-mediated process with high affinity for PMX. Potential alterations in protonation of RFC or the folate molecule as a function of pH do not result in changes in affinity constants for antifolates. Whereas both activities at low pH have similarities, they can be distinguished by folic acid and PT523, agents for which they have very different structural specificities.

Antimetabolites, Antineoplastic↗

PubMed1996

Comparison of methotrexate polyglutamylation in L1210 leukemia cells when influx is mediated by the reduced folate carrier or the folate receptor. Lack of evidence for influx route-specific effects.

We previously described a methotrexate-resistant L1210 cell line (MTXrA) that lacks a functional reduced folate carrier and does not appreciably express the folate receptor. In the present study, we utilized MTXrA cell lines stably transfected with cDNAs encoding either the folate receptor or the reduced folate carrier to investigate the influence of the route of folate influx on the rate and extent of methotrexate polyglutamylation. At an extracellular methotrexate concentration of 0.1 microM, influx in the folate receptor transfectant (MTXrA-TF1) and in the reduced folate carrier transfectant (MTXrA-R1) was equal and methotrexate polyglutamates accumulated at an identical rate, but the onset was delayed until dihydrofolate reductase was saturated with the monoglutamate (approxmately 3 hr). The onset of polyglutamate formation was immediate and identical among the lines in cells pretreated with the lipophilic dihydrofolate reductase inhibitor trimetrexate to block methotrexate binding to dihydrofolate reductase. The spectra of individual methotrexate polyglutamates that accumulated were similar, with the tetraglutamate present as the predominant form. A 100-fold higher methotrexate concentration was required to detect methotrexate uptake and polyglutamylation in the transport defective parent MTXrA line, demonstrating that diffusion or an unidentified low affinity route also supports polyglutamylation. Since the folate receptor and the reduced folate carrier achieve nearly identical rates of polyglutamylation despite very different mechanisms of methotrexate delivery, the data suggest that transport-mediated substrate channeling to folylpolyglutamate synthetase is unlikely to play a role in tetrahydrofolate metabolism. This study supports the notion that it is the intracellular concentration of methotrexate achieved within the cell that drives polyglutamylation irrespective of its route of entry.

Animals↗

PubMed2005

Rat reduced-folate carrier-1 is localized basolaterally in MDCK kidney epithelial cells and contributes to the secretory transport of methotrexate and fluoresceinated methotrexate.

The reduced-folate carrier (Rfc-1), previously also called methotrexate carrier-1 (MTX-1), was recently identified as accounting for approximately 30% of the methotrexate (Mtx) uptake into rat kidney slices. The localization of the carrier and its contribution to secretory or reabsorptive flux of the drug was therefore evaluated in polarized epithelial layers of Madin Darby canine kidney (MDCK) cells. Confocal laser scanning microscopy revealed that the HA-epitope-tagged protein was sorted to the basolateral side. In flux assays, the basolateral-to-apical transport of fluoresceinated methotrexate (FMTX) was two-fold higher than in the apical-to-basolateral direction across rat Rfc-1 transfected, but not mock-transfected, monolayers. The same observation was made for unlabeled Mtx. This secretory transport of FMTX was inhibited by an excess of 1 mM Mtx and was saturable and temperature-dependent. No differences in directional flux were observed for the pure fluorescein label. Removal of sodium resulted in a marked decrease of directional FMTX flux. The pH profile of the active transport component showed a trough around 6.5 and a maximum at acidic pH, as reported for uptake into Rfc-1-expressing cells. Thus, rat Rfc-1 is sorted to the basolateral side in polarized MDCK epithelial cells and mediates the secretion of Mtx, probably in co-operation with efflux proteins, such as multidrug resistance associated proteins, which are also expressed in these cells.

Animals↗

PubMed2005

No association between common polymorphisms in genes of folate and homocysteine metabolism and the risk of Down's syndrome among French mothers.

The cause of the non-disjunction leading to trisomy 21 remains unclear. Recent evidence has suggested that 5,10-methylenetetrahydrofolate reductase (MTHFR) and/or methionine synthase reductase (MTRR) might contribute to the maternal risk of trisomy 21. The purpose of the present study was to analyse these findings among the French population and to investigate whether common polymorphisms in genes of the folate and homocysteine pathway, including the MTHFR 677C > T, MTHFR 1298A > C, the methionine synthase (MTR) 2756A > G, the cystathionine beta-synthase (CBS) 844Ins68 and the reduced folate carrier (RFC-1) 80G > A polymorphisms, contribute to the risk of trisomy 21. The risk was studied by analysing independent and combined genotypes in 119 case mothers and 119 control mothers. The MTHFR 677T, MTHFR 1298C, MTR2756G, MTRR66G, CBSIns68+ and the RFC-1 80G allele frequencies were not significantly different among French case mothers, compared with control mothers. The risk of having a child with trisomy 21 did not appear to be linked to polymorphisms in genes associated with folate and homocysteine metabolism.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1997

Defective transport is a common mechanism of acquired methotrexate resistance in acute lymphocytic leukemia and is associated with decreased reduced folate carrier expression.

Methotrexate (MTX) transport was examined in 27 patients with untreated acute lymphocytic leukemia (ALL) and 31 patients with relapsed ALL using a previously described fluorescent MTX analog (PT430) displacement assay (Blood 80:1158, 1992). Only 13% of untreated patients were considered to have impaired MTX transport, whereas more than 70% of relapsed patients had evidence of impaired MTX transport. To further characterize the basis for this defect, Northern analyses for the reduced folate carrier (RFC) were performed on the RNA available from the leukemic blasts of 24 patients in whom MTX transport had been measured. Six of nine samples with impaired MTX transport had decreased RFC expression (one had no detectable RFC expression), while three had no decrease in RFC expression. None of 15 samples with normal MTX transport had decreased RFC expression. A reverse-transcriptase polymerase chain reaction (RT-PCR) assay was developed to quantitate RFC mRNA expression more accurately. Decreased RFC expression was demonstrated in six of the nine samples with impaired MTX transport, confirming the results obtained by Northern blot. These data indicate decreased RFC expression associated with impaired MTX transport is observed in relapsed ALL following treatment with MTX-containing therapy.

Adolescent↗

PubMed2006

Genetic and nutritional deficiencies in folate metabolism influence tumorigenicity in Apcmin/+ mice.

Epidemiological studies indicate that adequate dietary folate is protective against colon cancer, although mechanisms remain largely elusive. We investigated the effects of genetic disruptions of folate transport and metabolism and of dietary folate deficiency in a mouse model of colon cancer, the Apc(min/+) mouse. Apc(min/+) mice with heterozygous knockout of the gene for reduced folate carrier 1 (Rfc1(+/-)) developed significantly fewer adenomas compared to Rfc1(+/+)Apc(min/+) mice [30.3+/-4.6 vs. 60.4+/-9.4 on a control diet (CD) and 42.6+/-4.4 vs. 55.8+/-7.6 on a folate-deficient diet, respectively]. Rfc1(+/-)Apc(min/+) mice also carried a lower tumor load, an indicator of tumor size as well as of tumor number. In contrast, there were no differences in adenoma formation between Apc(min/+) mice carrying a knockout allele for methionine synthase (Mtr(+/-)), an enzyme that catalyzes folate-dependent homocysteine remethylation, and Mtr(+/+)Apc(min/+) mice. However, in both Mtr groups of mice, dietary folate deficiency significantly increased adenoma number (from 32.3+/-3.8 on a CD to 48.1+/-4.2 on a folate-deficient diet), increased plasma homocysteine, decreased global DNA methylation in preneoplastic intestines and increased apoptosis in tissues. There were no genotype-associated differences in these parameters in the Rfc1 group, suggesting that the protection conferred by Rfc1 deficiency is carried out through a different mechanism. In conclusion, genetic and nutritional disturbances in folate metabolism can have distinct influences on tumorigenesis in Apc(min/+) mice; altered levels of homocysteine, global DNA methylation and apoptosis may contribute mechanistically to dietary influence.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗